The present invention relates to a chromoatographic method for obtaining tetrodotoxin of high purity.
Tetrodotoxin is a nonprotein neurotoxin with potent activity. It is found in diverse animal species, including puffer fish, goby fish, newt, frogs and the blue-ringed octopus.
Tetrodotoxin (TTX) has a chemical formula of C11H17N3O8, and has a molecular weight of 319.28. The Merck Index, 10th Ed. (1983), states tetrodotoxin is the generic name for the compound octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-(1,3)dioxocino(6,5-d)-pyrimidine-4,7,10,11, 12-pentol, which has the following structure: 
TTX molecule consists of a perhydroquinozoline group with a guanidine substituent and six hydroxyl groups. Pure TTX is a white crystalline powder, odorless and tasteless. It turns black around 220xc2x0 C. without decomposition. TTX is soluble in acidic aqueous solution, but not soluble in organic solvents. The pKa (aqueous) of TTX is 8.76; it is thus a basic alkaloid. Strongly acidic aqueous solutions can decompose TTX, so usually TTX is dissolved in an aqueous solution of a weak organic acid. TTX is relatively thermally stable in neutral to weakly acidic solutions, but will be destroyed promptly in a strongly acidic or basic aqueous solution.
By HPLC, the toxin extracted from puffer fish has been determined to be a mixture of more than 10 analogs, among which tetrodotoxin is predominant, accounting for 70% to 80% of the mass of the extract. Three other major analogs are tetrodonic acid, 4-epi tetrodotoxin and 4-epi anhydrotetrodotoxin, which are only slightly different in chemical properties but significantly different in biological activities. For example, the toxicity of tetrodotoxin is 4500 mouse units/milligram; 4-epi tetrodotoxin, only 710 mouse units/milligram; 4-epi anhydrotetrodotoxin, only 92 mouse units/milligram. [1] A method to extract tetrodotoxin with high yield from the tissues of an organism (such as the puffer fish ovaries) is described in co-pending application Ser. No. 09/695,711 filed Oct. 25, 2000 (Attorney Docket No. 3519-01010P). The TTX obtained by this method typically has a purity of 80% or higher. The TTX product obtained by the method can be further purified to 96%, even 99% or higher, by way of the technology of the present invention. The present invention therefore provides material suitable for uses, such as pharmaceutical formulations, that might require tetrodotoxin of very high purity.
The refining of TTX from biological tissues or cell cultures involves two major tasks. The first is to remove from TTX those impurities that are not a toxin substance, that is, compounds other than TTX and its analogues. Thus, it is necessary to separate TTX from substances such as the residual amino acids, polypeptides, and proteins. These substances are so different in physical properties from TTX and its analogues that they can be easily removed by adding organic solvent or by dissolving TTX in, for example, acetic acid and then precipitating the TTX from a basic solution such as an ammonia solution. Copending application Ser. No. 09/695,711 filed Oct. 25, 2000 (Attorney Docket No. 3519-0101P) describes this process in detail.
The other task is to remove TTX analogues. Reverse phase ion-pairing HPLC, utilizing a phosphate buffer as the mobile phase, has been described as a method for separating TTX from related compounds.[3] It was reported in late 1990s that TTX, 4-epi-TTX and anhydro-4-epi-TTX can be separated using several types of columns. For example, Nakamura and Yasumoto [1] used a cation exchange column and citrate buffer. Yasumoto and Michishita [4] used a octyldecylsilane column with aqueous acetonitrile, heptaflurobutyric acid, NaOH system as the mobile phase.
The present invention resides in a method for chromatographic purification of TTX, preferably using high performance liquid chromatography (HPLC). The method employs an octadecylsilane reverse phase separation matrix and an aqueous alkyl sulphonate solution as the mobile phase.